Immunomodulating and antiinflammatory agent

ABSTRACT

In the present invention, pharmaceutical compositions containing a histamine-added gamma-globulin as an effective component are used as an immunomodulating agent, a suppressive agent for eosinophilia, and as an antiinflammatory agent. The immunomodulating action is unexpectedly different from the action of conventional immunosuppressive agents. Accordingly, the compositions are useful as a pharmaceutical agent for the therapy of diseases associated with an abnormal immune system such as chronic articular rheumatism, systemic lupus erythematosus, multiple sclerosis, etc. and various types of immunodeficiency syndromes. In addition, the pharmaceutical compositions exhibit suppressive action upon hypereosinophilicity. They may be used as a therapeutic agent for infectious diseases, parasitic diseases, respiratory diseases, autoimmune diseases and eosinophilia caused by malignant tumors. The compositions are excellent antiinflammatory agents.

FIELD OF THE INVENTION

The present invention relates to the use of a histamine-addedgamma-globulin as pharmaceuticals. More particularly, it relates to animmunomodulating agent, a suppressive agent to hypereosinophilicity andan antiinflammatory agent containing the histamine-added gamma-globulinas an effective component.

BACKGROUND OF THE INVENTION

Histamine-added gamma-globulin restores histamine fixing ability whichis lowered in patients suffering from allergy and asthma. It is used asan agent for nonspecific hyposensitizing therapy for bronchial asthma,allergic rhinitis and allergic skin diseases such as urticaria, chroniceczema, atopic dermatitis, etc. Histamine-added gamma-globulin alsoexhibits suppressive action to liberation of histamine. It does notexhibit side effects exhibited by antihistamines and adrenocorticalhormones used as symptomatic remedies. It has therefore been widely usedas a pharmaceutical agent with high safety.

The present inventors have found that histamine-added gamma-globulinexhibits unexpected pharmacological action including immunomodulatingaction, suppressive action towards hypereosinophilicity, andantiinflammatory action.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions which containhistamine-added gamma-globulin in pharmaceutically effective amountswhich are effective as an immunomodulating agent, a hypereosinophilicitysuppressive agent, and an antiinflammatory agent. The compositions maybe in the form of a liquid injection or a dry preparation which isdissolved upon use for injection. In embodiments of the presentinvention the compositions may be used for treatment of mammaliandiseases associated with an abnormal immune system such as chronicarticular rheumatism, systemic lupus erythematosus, multiple sclerosis,and immunodeficiency syndromes. Therapeutic treatment of mammalianinfectious diseases, parasitic diseases, respiratory diseases,autoimmune diseases and eosinophilia caused by a malignant tumor mayalso be achieved with the pharmaceutical compositions. Thehistamine-added gamma-globulin may be made by admixing from about 1 toabout 200 parts by weight gamma-globulin with about 0.01 to about 2parts by weight of a histamine component. In preferred embodiments, fromabout 5 to about 50 parts by weight gamma-globulin may be combined withabout 0.05 to about 0.5 parts by weight of a histamine component toobtain the histamine-added gamma-globulin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the potentiating action of histamine-addedgamma globulin to anti-TNP IgM antibody production.

FIG. 2 is a graph showing the potentiating action of histamine-addedgamma globulin to anti-INP IgG antibody production.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention histamine-added gamma-globulin is used inpharmaceutically effective amounts in immunomodulating agents,hypereosinophilicity suppressive agents, and antiinflammatory agents.The histamine-added gamma-globulin provides unexpectedly effectiveimmunomodulating action, inhibiting action to hypereosinophilicity, andantiinflammatory action in the pharmaceutical compositions. It may bemanufactured by mixing of a gamma-globulin component with a histaminecomponent to obtain a substantially homogeneous mixture. Humangamma-globulin for use in the present invention in treating humans maybe prepared from serum or from placenta plasma or the like byconventional means. The histamine component may be free histamine or apharmacologically-acceptable salt thereof such as hydrochloride,phosphate, picrate, etc., or mixtures thereof.

In embodiments of the present invention, the histamine-addedgamma-globulin may be made by admixing from about 1 to about 200 partsby weight gamma-globulin with about 0.01 to about 2 parts by weight of ahistamine component. In preferred embodiments, from about 5 to about 50parts by weight gamma-globulin may be combined with about 0.05 to about0.5 parts by weight of a histamine component to obtain thehistamine-added gamma-globulin. For example, in the manufacture of theproduct of the present invention, 100 to 200 mg (preferably 5 to 50 mg)of gamma-globulin may, for example, be mixed with 0.01 to 2 micrograms(preferably 0.05 to 0.5 microgram) of histamine component withconventional mixing means.

The histamine-added gamma-globulin products of the present invention areprimarily administered as injections. They can be made into apharmaceutical composition as an isotonic solution using distilled waterfor injection or a physiological saline solution. In its manufacture,one or more additives such as auxiliary solubilizers, isotonizingagents, stabilizers, buffers, preservatives, etc. may be used inpharmaceutically acceptable amounts in addition to the gamma-globulincomponent and the histamine component. Examples of the applicableadditives are citric acid, sodium benzoate, glycine, sodium sulfite,sodium bisulfite, sodium pyrosulfite, sodium thiosulfate, cysteinehydrochloride, phosphates, sodium ascorbate, sodium chloride, sodiumbicarbonate, etc., and mixtures thereof.

Further, the product of the present invention may be prepared as aninjectable preparation which is dissolved upon actual use. Thus, each ofthe components may be mixed in a dry state. In another embodiment, amixed solution may be filled in vials or the like followed byfreeze-drying. In the manufacture of the dry preparation for injection,one or more fillers such as glucose, mannitol and sorbitol may be addedin addition to the above-mentioned additives. An example of a drypreparation for injection is a histamine-added immunized human globulinpreparation.

The present invention is illustrated by the following examples:

EXAMPLE 1

Mice were used as experimental animals in the following pharmacologicaltests and, accordingly, mouse gamma-globulin was used in place of humangamma-globulin. Both types of histamine-added gamma-globulin may beproduced in the same manner. Thus, for the pharmacological tests onmice, a mouse gamma-globulin and histamine dihydrochloride weredissolved in distilled water in the following mixing ratios:

    ______________________________________                                        Product of the                                                                              Amount of Mouse                                                                           Amount of                                           Present Invention                                                                           gamma-Globulin                                                                            Histamine.2HCl                                      ______________________________________                                        HG50           5.3 mg     0.10 microgram                                      HG75          12.0 mg     0.15 microgram                                      HG90          28.8 mg     0.30 microgram                                      ______________________________________                                    

Each solution was stirred at room temperature for two hours,freeze-dried and, upon use, dissolved by adding a physiological salinesolution thereto.

Each of the HG50, HG75 and HG90 products prepared above exhibitedsignificant effects in all of the following pharmacological tests and,accordingly, only the results obtained for HG75 will be given asrepresentative thereof:

I. IMMUNOMODULATING ACTION

The immunomodulating action was measured using the production of anantibody specific to trinitrophenyl (TNP) and also a delayedTNP-specific hypersensitivity (DTH) reaction as targets.

(1) Preparation of Trinitrophenyl-Bonded Sheep Red Cells (TNP-SRBC).

Trinitrobenzenesulfonic acid (TNBS) was dissolved in a physiologicalsaline solution buffered with phosphoric acid to prepare a solution (40mg/7.0 ml; pH 7.2) and then 1 ml of sheep red cell pellets was droppedthereinto with stirring. The mixture was allowed to stand at roomtemperature with stirring for several times under a light-shieldingstate and washed with a physiological saline solution three times. Thenit was centrifuged at 3,000 rpm for five minutes and made into asolution of 5×10⁹ cells/ml using a physiological saline solution.

(2) Production of a TNP-Specific Antibody.

TNP-SRBC (10⁹ cells) was intraperitoneally administered to male BALB/cmice of six to eight weeks age and the anti-TNP antibody in serum wasmeasured by an enzymatic immunoassay (ELISA) using adinitrophenyl-bovine serum albumin (DNP-BSA). The result was that apotent antibody production of anti-TNP-IgM and anti-TNP-IgG was notedhaving a peak on the 4th to 6th days. In the case of BALB/c nude micehaving no thymus, production of antibodies of both types was rarelynoted.

(3) TNP-Specific DTH Reaction.

The mice were sensitized with TNP-SRBC as described in paragraph (2)above. On the 14th day, 0.025 ml of TNB (4.7 mg/ml) was injected intothe right hind paw to induce a TNP-specific DTH reaction. After 24 hoursand 48 hours from the induction, the thickness of both paws was measuredusing a dial gauge. The difference in the thickness between the rightand left paws was expressed as the intensity of the DTH reaction. Theresult was that, after 24 hours from the induction, a DTH reaction wasclearly noted. However, in the case of BALB/c nude mice, no DTH reactionwas observed at all. This measuring system for the DTH reaction can besubjected to further tests using the same mice as in the case of theantibody production system of paragraph (2) above.

(4) Measurement of the Action of the Tested Drugs.

The above-mentioned test system was used for checking the action of: (a)the histamine-added mouse gamma-globulin (150 mg/kg/day) (the product ofthe present invention), (b) cyclosporin A (100 mg/kg/day), (c)cyclophosphamide (100 mg/kg/day), (d) prednisolone (0.5 kg/kg/day) and(e) levamizole (5 mg/kg/day) towards the anti-TNP antibody productionand to the TNP-specific DTH reaction. The drugs were administered byhypodermic injection four days from the sensitization with TNP-SRBC.

The results for the anti-TNP antibody production system is given in FIG.1 and FIG. 2. The results for the TNP-specific DTH reaction system isgiven in Table 1.

In the test results, significant difference in the average values fromthe control was calculated by means of the Student's t-test and isexpressed with asterisks (*: p<0.05; **: p<0.01; ***: p<0.001).

                  TABLE 1                                                         ______________________________________                                        Tested          Swelling in Paws (×10.sup.-2 mm)                        Compound        After 24 Hrs.                                                                            After 48 Hrs.                                      ______________________________________                                        Not Sensitized  11.7 ± 3.8***                                                                         10.0 ± 2.8*                                     Control         49.2 ± 3.5                                                                            21.7 ± 3.8                                      Product of this 26.7 ± 4.4**                                                                          10.8 ± 2.4*                                     Invention                                                                     Cyclosporin     15.8 ± 2.0***                                                                         10.8 ± 2.7*                                     Cyclophosphamide                                                                              13.3 ± 2.1***                                                                         15.8 ± 2.4                                      Prednisolone    30.0 ± 5.6*                                                                            8.3 ± 3.3*                                     Levamizole      40.0 ± 6.5                                                                            20.0 ± 5.0                                      ______________________________________                                    

II. INHIBITORY ACTION TO HYPEREOSINOPHILICITY

The inhibitory action to hypereosinophilicity was measured using aragweed pollen antigen induced reaction and a platelet activating factor(PAF) reaction.

(1) Hypereosinophilic Model Induced by Ragweed Pollen Antigen.

A ragweed pollen extract (which was diluted to an extent of 1,000 timesusing a physiological saline solution) was hypodermically injected intofemale BALB/c mice of six to eight weeks age for sensitization at thedose of 0.1 ml on the initiation day and on the first day and 0.2 ml onthe sixth, eighth and fourteenth days. On the twentieth day, 0.2 ml of a1,000 times diluted ragweed antigen was intraperitoneally injected intothe mice to induce the reaction. On the 24th hour after the induction,the peritoneal exudate cells were recovered and subjected to a Giemsastaining and the total cell numbers, number of eosinophils, number ofneutrophils and number of mononuclear cells were counted. As a result,the number of the eosinophils peaked 24 hours after the induction. Inthe case of BALB/c nude mice having no T cells, no exudation toperitoneum was noted at all both in eosinophils and in neutrophils.

(2) Measurement of the Action of the Tested Pharmaceuticals.

The above-mentioned hypereosinophilic models were used for checking theaction towards the hypereosinophilicity by a hypodermic injection of theproduct of the present invention. Thus, histamine-added mousegamma-globulin, at a dose of 3 mg/mouse/day twice a week for three weekswas administered until the induced day. Further tests were conducted byadministering the corresponding amount of each of histamine(dihydrochloride) and mouse gamma-globulin which are the constitutingcomponents of the histamine-added mouse gamma-globulin. In addition,cyclosporin A, which is known as an immuno-suppressive agent, washypodermically administered at the dose of 100 mg/kg/day for three daysfrom two days prior to the induction until the induced day and theresults were used for comparison.

An example of the results is given in Table 2:

                  TABLE 2                                                         ______________________________________                                                       Number of Eosinophils Exudated to                              Tested Pharmaceuticals                                                                       Peritoneum (×10.sup.5 cells)                             ______________________________________                                        Not Sensitized 0.4 ± 0.1***                                                Control        3.7 ± 0.4                                                   Product of the Invention                                                                     0.5 ± 0.1***                                                Histamine      3.4 ± 0.6                                                   gamma-Globulin 3.2 ± 0.5                                                   Cyclosporin A  0.4 ± 0.2***                                                ______________________________________                                    

(3) Measurement of the Action of the Tested Pharmaceuticals UsingHypereosinophilic Model Induced by Platelet Activating Factor (PAF).

A PAF (5 micrograms/200 microliters) was intraperitoneally injected intofemale BALB/c mice of six to eight weeks age to induce the reaction.After 24 hours from the induction, the peritoneal exudate cells wererecovered and the number of the eosinophils were counted. The resultinghypereosinophilic model was used for checking the suppressive action ofthe product of the present invention (i.e., histamine-added mousegamma-globulin) against the eosinophilicity in the same manner asmentioned above.

An example of the results is given in Table 3:

                  TABLE 3                                                         ______________________________________                                                       Number of Exudated Eosinophils                                 Tested Pharmaceutical                                                                        to Peritoneum (×10.sup.5 cells)                          ______________________________________                                        Not Sensitized  0.3 ± 0.1**                                                Control        2.3 ± 0.5                                                   Product of the Invention                                                                      0.8 ± 0.1*                                                 Histamine      2.2 ± 0.8                                                   gamma-Globulin 1.5 ± 0.2                                                   ______________________________________                                    

It is clear from the results of FIG. 1 and FIG. 2 that thehistamine-added gamma-globulin (the product of the present invention)exhibited significant promoting action upon IgG and IgM antibodyproduction. However, cyclosporin A and cyclophosphamide(immunosuppressive agents) significantly suppressed the production ofboth antibodies. Prednisolone (an adrenocortical hormone) and levemizole(said to exhibit an immunomodulating action) did not significantlyaffect antibody production at the dose of the present test.

On the other hand, as shown in Table 1 the product of the presentinvention exhibited a significant suppressive action to the delayed typehypersensitivity (DTH). Both cyclosporin A and cyclophosphamide showedclear suppressive actions. In addition, prednisolone showed a weaksuppressive action while levemisole had no significant effect.

As such, cyclosporin A and cyclophosphamide (conventionalimmunosuppressive agents) significantly suppressed the immunoreactionsof both IgG and IgM antibody production and the DTH reaction. However,histamine-added gamma-globulin (the product of the present invention)exhibited a promoting action towards the antibody production and asuppressive action upon the DTH reaction. As such, the histamine-addedgamma-globulin has an immunomodulating action which is clearlyunexpectedly different from the action of conventional immunosuppressiveagents.

Furthermore, as shown in Table 2, the product of the present inventionsignificantly suppressed the eosinophil exudation into peritoneum in thehypereosinophilic model induced by a ragweed pollen antigen. Moreover,as apparent from the results of Table 3, the histamine-addedgamma-globulin exhibited a clear suppressive action upon thehypereosinophilicity induced by administration of PAF instead of by anantigen induction like cyclosporin A which is an immunosuppressiveagent. It is clear that said suppressive action uponhypereosinophilicity is an action which is specific to the product ofthe present invention because said action is not observed for each ofthe histamine and the gamma-globulin which are the components of thehistamine-added gamma-globulin. Moreover, the product showed asignificant suppressive action to the above-mentioned antigen-inducedand PAF-induced hypereosinophilic models even in the same administrationterms as for cyclosporin A (i.e., from two days before the inductionuntil the induced day).

It is clear from the above-mentioned results of the pharmacologicaltests that the pharmaceutical composition of the present invention has aspecific immunomodulating action which is unexpectedly different fromthe action of conventional immunosuppressive agents. Accordingly, saidpharmaceutical composition is useful as a pharmaceutical agent for thetherapy of diseases associated with an abnormal immune system such aschronic articular rheumatism, systemic lupus erythematosus, multiplesclerosis, etc. and various types of immunodeficiency syndromes. Inaddition, since the product of the present invention exhibits asuppressive action upon hypereosinophilicity, it can be used as atherapeutic agent for infectious diseases, parasitic diseases,respiratory diseases, autoimmune diseases and eosinophilia caused bymalignant tumor, etc.

Eosinophils are known as effector cells which accumulate at thestimulated portion which causes inflammation and results in inflammatorysymptoms. Accordingly, the agents which suppress the increase ineosinophils can be used as remedies for suppressing inflammation. Thepharmaceutical composition of the present invention containing ahistamine-added gamma-globulin exhibits an action of suppressing thetumor in the DTH reaction in addition to the above-mentioned suppressiveaction upon an increase in eosinophils. Accordingly, the composition isquite useful as an excellent antiinflammatory agent as well.

EXAMPLE 2

An example of a formulation of a pharmaceutical composition fortreatment of humans according to the present invention is:

    ______________________________________                                                                    Amount                                            Formulation                                                                              Components       In a Vial                                         ______________________________________                                        Injection  Histamine-Added Human                                                                          36 mg                                             (2 ml)     gamma-Globulin                                                                Histamine         0.45 microgram                                              Dihydrochloride                                                               Sodium Chloride  q.s.                                              ______________________________________                                    

The dose may be suitably selected depending upon the type of thedisease, degree of the disease, age and sex of the patient, term of theadministration, etc. to provide a pharmaceutically effective amount ofthe histamine-added human gamma globulin.

What is claimed is:
 1. A method of treating an autoimmune disease exceptfor autoimmune allergic skin diseases comprising administering apharmaceutically effective amount of histamine-added gamma-globulin. 2.A method as claimed in claim 1 wherein the disease is multiplesclerosis.
 3. A method as claimed in claim 1 wherein the disease issystemic lupus erythematosus.
 4. A method as claimed in claim 1 whereinthe disease is chronic articular rheumatism.
 5. A method as claimed inclaim 1 wherein said histamine-added gamma-globulin is obtained byadmixing from about 1 to about 200 parts by weight of a gamma-globulincomponent with about 0.01 to about 2 parts by weight of a histaminecomponent.
 6. A method as claimed in claim 5 wherein saidhistamine-added gamma-globulin is obtained by admixing from about 5 toabout 50 parts by weight of a gamma-globulin component with about 0.05to about 0.5 parts by weight of a histamine component.
 7. A method asclaimed in claim 5 wherein said gamma-globulin component is humangamma-globulin.
 8. A method as claimed in claim 5 wherein said histaminecomponent is at least one pharmaceutically acceptable histamine salt. 9.A method as claimed in claim 8 wherein said salt is histaminedihydrochloride.
 10. A method as claimed in claim 1 wherein saidhistamine-added gamma-globulin is formulated as a dry preparation forinjection.
 11. A method as claimed in claim 1 wherein saidhistamine-added gamma-globulin is solubilized for injection.